It has been suggested to use O.sup.6 -alkyl guanine derivatives possessing O.sup.6 -alkylguanine-DNA alkyltransferase depleting activity in order to enhance the effectiveness of chemotherapeutic alkylating agents, principally those that methylate or chloroethylate DNA, used for killing tumour cells. There is increasing evidence that in mammalian cells the toxic and mutagenic effects of alkylating agents are to a large extent a consequence of alkylation at the O.sup.6 -position of guanine in DNA. The repair of O.sup.6 -alkylguanine is mediated by ATase, a repair protein that acts on the O.sup.6 -alkylated guanine residues by stoichiometric transfer of the alkyl group to a cysteine residue at the active site of the repair protein in an autoinactivating process. The importance of ATase in protecting cells against the biological effects of alkylating agents has been most clearly demonstrated by the transfer and expression of cloned ATase genes or cDNAs into ATase deficient cells: this confers resistance to a variety of agents, principally those that methylate or chloroethylate DNA. Whilst details of the mechanism of cell killing by O.sup.6 -methylguanine in ATase deficient cells is not yet clear, killing by O.sup.6 -chloroethylguanine occurs through DNA interstand crosslink formation to a cytosine residue on the opposite strand via a cyclic enthanoguanine intermediate, a process that is prevented by ATase-mediated chloroethyl group removal or complex formation.
The use of O.sup.6 -methylguanine and O.sup.6 -n-butylguanine for depleting ATase activity has been investigated (Dolan et al., Cancer Res., (1986) 46, pp. 4500; Dolan et al., Cancer Chemother. Pharmacol., (1989) 25, pp 103. O.sup.6 -benzylguanine derivatives have been proposed for depleting ATase activity in order to render ATase expressing cells more susceptible to the cytotoxic effects of chloroethylating agents (Moschel et al., J. Med. Chem., 1992, 35, 4486). U.S. Pat. No. 5,091,430 and International Patent Application No. WO 91/13898 Moschel et al. disclose a method for depleting levels of O.sup.6 -alkylguanine-DNA alkyl-transferase in tumour cells in a host which comprises administering to the host an effective amount of a composition containing O.sup.6 -benzylated guanine derivatives of the following formula: ##STR2## wherein Z is hydrogen, or ##STR3## and R.sup.a is a benzyl group or a substituted benzyl group. A benzyl group may be substituted at the ortho, meta or para position with a substituent group such as halogen, nitro, aryl such as phenyl or substituted phenyl, alkyl of 1-4 carbon atoms, alkoxy of 1-4 carbon atoms, alkenyl of up to 4 carbon atoms, alkynyl of up to 4 carbon atoms, amino, monoalkylamino, dialkylamino, trifluoromethyl, hydroxy, hydroxymethyl, and SO.sub.n R.sup.b wherein n is 0, 1, 2 or 3 and R.sup.b is hydrogen, alkyl of 1-4 carbon atoms or aryl. Chae et al., J. Med. Chem., 1994, 37, 342-347 describes tests on O.sup.6 -benzylguanine analogs bearing increasingly bulky substituent groups on the benzene ring or at position 9. Chae et. al., J. Med. Chem. 1995, 38, 359-365 describe several 8-substituted O.sup.6 -benzylguanines, 2- and/or 8-substituted 6-(benzyloxy)purines, substituted 6(4)-(benzyloxy)pyrimidines, and a 6-(benzyloxy)-s-triazine which were tested for their ability to inactivate ATase. Two types of compounds were identified as being significantly more effective than O.sup.6 -benzylguanine at inactivating ATase in human HT29 colon tumour cell extracts. These were 8-substituted O.sup.6 -benzylguanines bearing electron-withdrawing groups at the 8-position (e.g. 8-aza-O.sup.6 -benzylguanine and O.sup.6 -benzyl-8-bromoguanine) and 5-substituted 2,4-diamino-6-(benzyloxy)pyrimidines bearing electron withdrawing groups at the 5-position (e.g. 2,4-diamino-6-(benzyloxy)-5-nitroso- and 2,4-diamino-6-(benzyloxy)-5-nitropyrimidine). The latter derivatives were also more effective than O.sup.6 -benzylguanine at inactivating ATase in intact HT29 colon tumour cells. WO 96/04280 published after the priority dates of this application concerns similar substituted O.sup.6 -benzylguanines and 6(4)-benzyloxypyrimidines.
The present Applicants are also Applicants in International Patent Application PCT/IE94/00031 which was published under No. WO 94/29312. U.S. patent application Ser. No. 08/568,576, filed Dec. 7, 1995, is the corresponding application in the United States (the contents of which are incorporated herein by reference in their entirety) described O.sup.6 -substituted guanine derivatives of formula I: ##STR4## wherein Y is H, ribosyl, deoxyribosyl, or ##STR5## wherein X is O or S, R" and R'" are alkyl, or substituted derivatives thereof;
R' is H, alkyl or hydroxyalkyl; PA1 R is (i) a cyclic group having at least one 5- or 6-membered heterocyclic ring, optionally with a carbocyclic or heterocyclic ring fused thereto, the or each heterocyclic ring having at least one hetero atom chosen from O, N, or S, or a substituted derivative thereof; or PA1 R.sup.2 is selected from H, C.sub.1 -C.sub.5 alkyl, halogen or NH.sub.2, PA1 R.sup.4 and R.sup.5 which are the same or different are selected from H, NH--Y' or NO.sub.n wherein Y' is H, ribosyl, deoxyribosyl, arabinosyl, R"XCHR"' wherein X is O or S and R" is alkyl and R"' is H or alkyl, or substituted derivatives thereof, PA1 n=1 or 2, PA1 or R.sup.4 and R.sup.5 together with the pyrimidine ring form a 5- or 6-membered ring structure containing one or more hetero atoms, PA1 with the proviso that R.sup.2 is not NH.sup.2 if R.sup.4 and R.sup.5 form a ring structure IX ##STR7## wherein Y is H, ribosyl, deoxyribosyl, or ##STR8## wherein X is O or S, R" and R"' are alkyl, or substituted derivatives thereof, PA1 and with the proviso that R is not phenyl in the following circumstances a) to h): PA1 Y' is as defined for formula II above, PA1 R.sup.6 is a cyclic group having at least one 5- or 6-membered heterocyclic ring, optionally with a carbocyclic or heterocyclic ring fused thereto, the or each heterocyclic ring having at least one hetero atom chosen from O, N or S, or a substituted derivative thereof, PA1 Y' is as defined for formula II. PA1 Y' is as defined for formula XIII, preferably Y' is H or HOCH.sub.2 CH.sub.2 OCH.sub.2 --; PA1 R.sup.2 is H, NH.sub.2, C.sub.1 -C.sub.5 alkyl, preferably methyl, or halogen, preferably fluorine; PA1 R.sup.3 is H or OH: PA1 Y' is as defined for formula XIII; PA1 X is CH or N; PA1 A is CH or N; and preferably when X.dbd.N, A.dbd.CH PA1 X is CH or N PA1 A is CH or N; PA1 Z is O or S or CH.dbd.CH PA1 U is CH or N; PA1 V is CH or N; PA1 W is CH or N; PA1 T is H, NH.sub.2 or NO.sub.n where n=1 or 2; PA1 Q is H, NH.sub.2 or NO.sub.n where N=1 or 2; PA1 Y' is as defined for formula II
(ii) naphthyl or a substituted derivative thereof; PA2 (ii) phenyl or a substituted derivative thereof,
and pharmaceutically acceptable salts thereof.
In order to be useful for depleting ATase activity and thus enhance the effects of the above-mentioned chemotherapeutic agents, compounds should have combination of characteristics assessed by reference to:
1) In vitro inactivation of recombinant ATase. PA0 2) Stability. PA0 3) Solubility. PA0 4) Inactivation of ATase in mammalian cells and/or tumour xenografts. PA0 5) Sensitization of mammalian cells and/or tumour xenografts to the killing or growth inhibitory effects of the said chemotherapeutic agents. PA0 a) if R.sup.2 and R.sup.5 are NH.sub.2 and R.sup.4 is NO or NO.sub.2 PA0 b) if R.sup.2 is NH.sub.2 and R.sup.4 and R.sup.5 form a ring structure X ##STR9## c) if R.sup.2 is NH.sub.2 and R.sup.4 and R.sup.5 form a ring structure XI ##STR10## d) if R.sup.2 is NH.sub.2, and R.sup.4 is NO.sub.2 and R.sup.5 is H or CH.sub.3 PA0 e) if R.sup.2, R.sup.4 and R.sup.5 are NH.sub.2, PA0 f) if R.sup.2 and R.sup.5 are NH.sub.2 and R.sup.4 is H, PA0 g) if R.sup.2 is H, and R.sup.4 is NO.sub.2 and R.sup.5 is NH.sub.2, or PA0 h) if R.sup.2 is F or OH, and R.sup.4 and R.sup.5 form a ring structure XII ##STR11##
The behaviour of novel compounds in this combination of tests is unpredictable. Molecular interactions including steric factors in the unpredictability of ATase inactivation may be related to the nature of the environment of the cysteine acceptor site in the ATase molecule.
The structure of the ATase protein derived from E. coli (Ada gene) has been elucidated by X-ray crystallographic techniques (M. H. Moore et. al., EMBO Journal, 1994, 13, 1495.). While the amino acid sequence of human ATase differs somewhat from that of bacterial origin, all known ATases (human, rodent, yeast, bacterial) contain the cysteine (Cys) acceptor site in a common fragment, Pro-Cys-His-Arg. A homology model of human ATase generated by computer from the crystal structure of the Ada protein (J. E. A. Wibley et. al., Anti-Cancer Drug Design, 1995, 10, 75.) resembles it in having the Cys acceptor buried in a pocket deep in the protein. Considerable distortion of the structure is necessary to bring either an O.sup.6 -alkylated guanine residue in intact DNA, or even free guanine alkylated by a relatively large group like benzyl, close to the Cys acceptor. These configurational changes are initiated by a characteristic binding of duplex DNA to the protein (K. Goodtzova et. al. Biochemistry, 1994, 33, 8385).
Since the amino acid components and dimensions of the ATase active site "pocket" are still unknown as are the details of the mechanism involved, it is impossible to predict the activity of a particular O.sup.6 -alkylated guanine or analogous ring system.
Published work in this field relates predominantly to the use of O.sup.6 -alkyl guanine derivatives having a nucleus identical to that of guanine in DNA. Chae et. al., J. Med. Chem. 1995, 38, 359-365 have described tests on a limited number of compounds in which the guanine ring was modified. However these compounds all had benzyl substitution at the O.sup.6 -position of the modified guanine ring or 6(4)-benzyloxy substitution on the pyrimidine ring. The observation that subtle changes in the substituents on the guanine ring or in the purine skeleton can generate agents that are very ineffective ATase inactivators, in comparison with their "parent" structure, suggests that more substantial modifications might also disrupt the ATase inactivating function.
There is a need for additional novel compounds useful for depleting ATase activity in order to enhance the effects of chemotherapeutic agents such as chloroethylating or methylating anti-tumour agents. It is a further object to provide compounds having better ATase inactivating characteristics than O.sup.6 -benzylguanine and having different solubility patterns.
Another object of the invention is to provide pharmaceutical compositions containing compounds which are useful for depleting ATase activity. A further object of the present invention is to provide a method for depleting ATase activity in tumour cells. A still further object of the invention is to provide a method for treating tumour cells in a host in such a way that they become more sensitive to the above-mentioned alkylating agents.
The present invention provide 6-hetarylalkyloxy pyrimidine derivatives of formula II: ##STR6## wherein R is (i) a cyclic group having at least one 5- 6-membered heterocyclic ring, optionally with a carbocyclic or heterocyclic ring fused thereto, the or each heterocyclic ring having at least one hetero atom chosen from O, N or S, or a substituted derivative thereof; or
and pharmaceutically acceptable salts thereof,
Certain O.sup.6 -substituted guanine derivatives within the scope of the general formula in WO 94/29312 but not published therein have been found to have a surprisingly advantageous combination of properties which justifies the selection of such derivatives from among the class defined in WO 94/29312.
In another aspect, the present invention provides guanine derivatives of formula XIII: ##STR12## wherein E is O or S,
and pharmaceutically acceptable salts thereof, with the proviso that compounds published in WO 94/29312 are disclaimed.
In particular, the present invention selects advantageous compounds of formula XIV: ##STR13## wherein R.sup.10 is bromo, chloro or cyano, and
Most preferably, R.sup.10 is bromo. A particularly preferred and selected compound is O.sup.6 -(4-bromothenyl)guanine having the formula XV: ##STR14##
This compound has an advantageous combination of properties including potential for oral administration.
R or R.sup.6 may suitably be a 5- or 6-membered heterocyclic ring or a benzo derivative thereof, in which latter case the pyrimidine moiety may be attached to R or R.sup.6 at either the heterocyclic or the benzene ring.
In preferred embodiments, R or R.sup.6 is a 5-membered ring containing S or O, with or without a second ring fused thereto.
Preferably, R or R.sup.6 is a heterocyclic ring having at least one S atom; more preferably, R or R.sup.6 is a 5-membered heterocyclic ring having at least one S atom; and most preferably, R or R.sup.6 is a thiophene ring or a substituted derivative thereof. Alternatively, R or R.sup.6 may be a heterocyclic ring having at least one O atom, particularly, a 5-membered heterocyclic ring having at least one O atom and more particularly R or R.sup.6 may be a furan ring or a substituted derivative thereof. As another alternative, R or R.sup.6 may be a heterocyclic ring having at least one N atom, particularly R or R.sup.6 may be a 6-membered heterocyclic ring having at least one N atom and in particular, R or R.sup.6 may be a pyridine ring.
The carbocyclic or heterocyclic ring fused to the heterocyclic ring in R or R.sup.6 may itself be bicyclic e.g. naphthalene.
In general the term "substituted derivative" as used in relation to any of the compounds of the invention means any substituted derivative whose presence in the compound is consistent with the compound having ATase depleting activity.
In the definition of Y or Y', the term "substituted derivative" includes further substitution by one or more of the following groups: hydroxy, halo, alkoxy, amino, alkylamino, amido or ureido. In a particularly preferred group of compounds, R" is hydroxy-substituted alkyl and R"' is H, so that Y' is hydroxyalkoxymethyl, preferably having 1 to 10 carbon atoms in the alkoxy group.
In the definition of R or R.sup.6, the term "substituted derivative" includes substitution of the heterocyclic ring(s) and/or carbocyclic ring(s) by one or more of the following groups: alkyl, alkenyl, alkynyl, alkoxy, aryl, halo, haloalkyl, nitro, cyano, azido, hydroxyalkyl, SO.sub.n R.sup.7 where R.sup.7 is alkyl and n=0, 1 or 2, or a carboxyl or ester group of the formula --COOR.sup.8 wherein R.sup.8 is H or alkyl. Halo, haloalkyl, cyano, alkylenedioxy, SO.sub.n R.sup.7 (as defined above) and --COOR.sup.8 wherein R.sup.8 is alkyl are preferred substituents.
An alkyl, alkoxy, alkenyl, or alkynyl group preferably contains from 1 to 20, more preferably from 1 to 10 and most preferably from 1 to 5 carbon atoms. Halo includes iodo, bromo, chloro or fluoro. An aryl group preferably contains from 1 to 20, more preferably from 1 to 10 carbon atoms, particularly 5 or 6 carbon atoms.
One embodiment of the invention provides a pharmaceutical composition containing compounds of formula II or formula XIII, as defined above, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. Optionally the composition may also contain an alkylating agent such as a chloroethylating or methylating agent.
In a further embodiment, the present invention provides a method for depleting ATase activity in a host comprising administering to the host an effective amount of a composition containing a compound of formula II or formula XIII as defined above, or a pharmaceutically acceptable salt thereof, more particularly a pharmaceutical composition as defined above. This method may alternatively be defined as a method of depleting ATase mediated DNA repair activity in a host.
The invention further provides a method for treating tumour cells in a host comprising administering to the host an effective amount of a composition containing a compound of formula II or formula XIII as defined above or a pharmaceutically acceptable salt thereof, more particularly a pharmaceutical composition as defined above and administering to the host an effective amount of a composition containing an alkylating agent. The method may be used for treatment of neoplasms including those which are known to be sensitive to the action of alkylating agents e.g. melanoma and glioma and others whose resistance to treatment with alkylating agents alone may be overcome by the use of an inactivator according to the invention.
The term "pharmaceutically acceptable salts" as used in this description and the claims means salts of the kind known in the pharmaceutical industry including salts with inorganic acids such as sulfuric, hydrobromic, nitric, phosphoric or hydrochloric acid and salts with organic acids such as acetic, citric, maleic, fumaric, benzoic, succinic, tartaric, propionic, hexamoic, heptanoic, cyclopentanepropionic, glycolic, pyruvic, lactic, malonic, malic, o-(4-hydroxy-benzoyl)benzoic, cinnamic, mandelic, methanesulfonic, ethanesulfonic, 1,2-ethanedisulfonic, 2-hydroxyethanesulfonic, benzenesulfonic, p-chlorobenzenesulfonic 2-naphthalenesulfonic, p-toluenesulfonic, camphorsulfonic, 4-methyl-bicyclo[2.2.2]oct-2-ene-1-carboxylic, glucoheptonic, 4,4'-methylenebis(3-hydroxy-2-naphthoic), 3-phenylpropionic, trimethyl-acetic, tertiary butylacetic, lauryl sulfuric, gluconic, glutamic, hydroxynaphthoic, salicyclic, stearic, or muconic, and the like.
Subject to the provisos above the preferred compounds of the invention are those of: